The broad, long-term objectives are to throw further light on the biochemistry and biological significance of the phosphoinositide signalling system. More specifically, (1) What is the role of inositol 1,2-cyclic 4,5-trisphosphate (cIP3) in the phosphoinositde signalling process? Paramount on the list will be a search for a cIP3 receptor unique from the inositol (1,4,5)-trisphosphate (I(1,4,5)P3) receptor because the discovery of such a receptor would provide very strong evidence for a second messenger function for cIP3. Possible second messenger or regulatory roles will be tested in permeabilized acini and cell-free fractions, such as (a) inhibition of binding of I(1,4,5)P3 at its receptor. This could dampen Ca2+ mobilization after its initial spike; (b) synergism between cIP3 and I(1,4,5)P3 or I(1,3,4,5)P3 on Ca2+ influx; (c) activation by cIP3 of PI hydrolysis in the endoplasmic reticulum (ER). (2) What is the function of free arachidonic acid (AA) and metabolites in agonist-stimulated cells? We will examine the capability of AA to release Ca2+ from permeabilized and unpermeabilized pancreatic acinar cells. We will also study the activation of protein kinase C (PKC) by AA alone or in combination with activators of PKC, such as Ca2+, diacylglycerol (DAG), and phosphatidylserine. (3) Is there direct phosphodiesteratic cleavage of PI in the ER, or is the agonist-stimulated loss of PI strictly by phosphorylation of PI to form PIP and then PIP2 in the plasma membrane (PM)? Microsomes derived from the ER will be incubated with cIP3, I(1,4,5)P3, DAG, Ca2+ etc., which may be messengers which could trigger direct PI breakdown. Also, direct breakdown could be established if we are able to show a formation of I(l)P in excess of that which could be formed by the I(1,4,5)P3 degradation pathway. (4) We will study the formation of cIP3 and AA in the central nervous system. We will concentrate mainly on the cerebellum, which has the highest density of IP3 receptors and which releases substantial amounts of prostaglandins in response to low concentrations of acetylcholine. (5) We are initiating molecular-biological studies on the phosphoinositide signalling system. We have essentially purified the 53 Kd PIP-kinase band on SDS-PAGE from human red cells and are preparing antibodies to screen a human brain cDNA library in the expression vector, lambda GT11.